Akt2 (IC50 = 17 nM); p70S6K (IC50 = 85 nM); Akt1 (IC50 = 32 nM); Akt3 (IC50 = 47 nM); PKA (IC50 = 20 nM)
AT7867 is a dual inhibitor of the Akt kinase family and p70 S6 kinase (p70 S6K), key regulators of the PI3K/mTOR signaling pathway. In recombinant enzyme assays, it exhibits IC50 values of 16 nM for Akt1, 35 nM for Akt2, 21 nM for Akt3, and 26 nM for p70 S6K. It shows minimal cross-reactivity with other kinases (e.g., PKA, EGFR, VEGFR2) with IC50 values > 1000 nM [1]

The inhibition of AKT2 by AT7867 is shown to be ATP-competitive with a Ki of 18nM. Although AT7867 exhibits a pronounced window of selectivity against kinases from other kinase sub-families, it also exhibits potent activity against the structurally related AGC kinases p70S6K and PKA. Studies on the inhibition of in vitro growth have revealed that AT7867 inhibits proliferation in a number of human cancer cell lines. AT7867 appears to be least effective in the two prostate lines tested (IC50 values range from 10–12 M) and most potent at inhibiting proliferation in the MES-SA uterine, MDA-MB-468 and MCF-7 breast, HCT116 and HT29 colon lines (IC50 values range from 0.9–3 M)[1].

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In human prostate cancer cell lines (PC-3, LNCaP) with activated PI3K/Akt signaling, AT7867 (0.01-10 μM) inhibited cell proliferation in a dose-dependent manner. The IC50 values were 0.28 μM for PC-3 cells and 0.42 μM for LNCaP cells after 72 hours of treatment (SRB assay). Western blot analysis showed that 0.5 μM AT7867 reduced phosphorylation of Akt (Ser473/Thr308) by > 90%, p70 S6K (Thr389) by 85%, and downstream target S6 ribosomal protein (Ser235/236) by 80% within 24 hours, while total protein levels of these kinases remained unchanged [1]
- In human ovarian cancer cell line SKOV3 (with constitutive Akt activation), AT7867 (0.1-5 μM) induced dose-dependent apoptosis after 48 hours. Flow cytometry with Annexin V-FITC/PI staining revealed that 2 μM AT7867 increased the apoptotic rate from 3% (control) to 36%. Additionally, 1 μM AT7867 inhibited colony formation of SKOV3 cells by 70% compared to vehicle control (crystal violet staining, 14-day culture) [1]
- In PC-3 cells co-treated with AT7867 (0.2 μM) and docetaxel (5 nM), the combination showed synergistic antiproliferative activity with a combination index (CI) of 0.55 (CI < 1 indicates synergy). The apoptotic rate in the combination group was 42%, significantly higher than 17% (AT7867 alone) and 14% (docetaxel alone) [1]

Following oral administration at 20 mg/kg, the elimination of AT7867 from plasma appears to be similar to that observed after i.v. administration. Following an oral dose of 20 mg/kg of AT7867, plasma levels stay above 0.5 M for at least 6 hours. The bioavailability through the oral route is calculated to be 44% under the assumption that linear pharmacokinetics occur after intravenous administration. Therefore, this model is used for in vivo pharmacodynamic (PD) biomarker studies. Athymic mice with MES-SA tumors are given doses of AT7867 (90 mg/kg p.o. or 20 mg/kg i.p.) after pharmacokinetic and tolerability studies, and the phosphorylation status of GSK3 and S6RP in tumors is tracked over time. At 2 and 6 hours after AT7867 treatment, a definite inhibition of the phosphorylation of the two markers of pathway activity is observed. Total levels of GSK3 and S6RP are significantly decreased by 24 hours[1].

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In a nude mouse xenograft model of human prostate cancer (PC-3), AT7867 was administered orally at doses of 25 mg/kg and 50 mg/kg once daily for 21 days. Compared to the vehicle control (0.5% carboxymethyl cellulose sodium + 0.1% Tween 80), the 25 mg/kg group showed a 52% reduction in tumor volume, and the 50 mg/kg group showed a 75% reduction. Immunohistochemical staining of tumor tissues demonstrated decreased p-Akt (Ser473) expression (-85%) and Ki-67 (proliferation marker) positive cells (-60%) in the 50 mg/kg group [1]
- In a nude mouse xenograft model of human ovarian cancer (SKOV3), AT7867 was administered orally at 50 mg/kg once daily for 14 days. The treatment resulted in a 68% reduction in tumor weight compared to the vehicle control. Western blot analysis of tumor lysates confirmed reduced p-p70 S6K (Thr389) and increased cleaved caspase-3 (apoptosis marker) [1]
- In the PC-3 xenograft model, combination treatment with AT7867 (25 mg/kg oral, once daily) and docetaxel (10 mg/kg i.p., once weekly) for 21 days resulted in an 88% reduction in tumor volume, which was significantly greater than monotherapy effects (52% for AT7867 alone, 45% for docetaxel alone) [1]

AKT2, PKA, p70S6K, and CDK2/cyclinA kinase assays are all conducted using a radiometric filter binding format. Reactions for the assay are set up with the compound present. For AKT2, the AKT2 enzyme is incubated for 4 hours with 25 mM AKTide-2T peptide (HARKRERTYSFGHHA), 20 mM MOPS, pH 7.2, 5 mM EDTA, 15 mM MgCl2, 1 mM sodium orthovanadate, 1 mM DTT, 10 g/mL BSA, and 30 mM ATP (1.16 Ci/mmol). For PKA, 50 mM of the peptide (GRTGRRNSI) and the PKA enzyme are incubated for 20 minutes in a solution containing 2 mM MOPS, pH 7.2, 25 mM -glycerophosphate, 5 mM EDTA, 15 mM MgCl2, 1 mM orthovanadate, 1 mM DTT, and 40 mM ATP (0.88 Ci/mmol). For p70S6K, the enzyme and 25 M peptide substrate (AKRRRLSSLRA) are incubated for 60 minutes in 10 mM MOPS, pH 7, 0.2 mM EDTA, 1 mM MgCl2, 0.01% β-mercaptoethanol, 0.1 mg/mL BSA, 0.001% Brij-35, 0.5% glycerol, and 15μM ATP (2.3 Ci/mmol). For CDK2, the CDK2/cyclinA enzyme and 0.12 g/ml histone H1 are incubated for 4 hours in 20 mM MOPS, pH 7.2, 25 mM β-glycerophosphate, 5 mM EDTA, 15 mM MgCl2, 1 mM sodium orthovanadate, 1 mM DTT, 0.1 mg/ml BSA and 45 μM ATP (0.78 Ci/mmol) for 4 hours. Orthophosphoric acid is used to stop assay reactions, and the stopped reaction mixture is then transferred to Millipore MAPH filter plates where it is filtered. The plates are then washed, scintillant added and radioactivity measured by scintillation counting on a Packard TopCount. IC50 values are calculated from replicate curves using GraphPad Prism software. AKT1 and 3 enzyme assays are carried out, while all other enzyme assays are performed[1].

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Akt1 Kinase Inhibition Assay: Recombinant human Akt1 (0.1 μg per reaction) was mixed with 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 10 μM ATP (including [γ-32P]ATP), 20 μM Crosstide (Akt-specific substrate peptide), and serial dilutions of AT7867 (1 nM-500 nM) in a total volume of 50 μL. The reaction mixture was incubated at 30°C for 30 minutes, then terminated by adding 25 μL of 30% trichloroacetic acid. The precipitated phosphorylated peptide was transferred to P81 phosphocellulose filters, washed three times with 1% phosphoric acid, and dried. Radioactivity was measured using a liquid scintillation counter, and IC50 was calculated via four-parameter logistic regression [1]
- p70 S6K Kinase Assay: Recombinant human p70 S6K (0.2 μg per reaction) was incubated with 25 mM HEPES (pH 7.4), 10 mM MgCl2, 1 mM EGTA, 200 μM ATP (including [γ-32P]ATP), 1 μg/mL 40S ribosomal subunit (p70 S6K substrate), and AT7867 (5 nM-1000 nM) for 45 minutes at 37°C. The reaction was terminated with SDS sample buffer, and phosphorylated 40S subunit was separated by 12% SDS-PAGE. The gel was dried, and radioactivity was detected by autoradiography. IC50 was determined by plotting the percentage of remaining kinase activity against drug concentration [1]

Prior to AT7867 treatment, cells are grown for 24 hours in 10% FBS-supplemented medium in 96-well microplates at a density of 16,000 cells per well. The cells are supplemented for an hour with AT7867 or vehicle control. A phospho-GSK3 (serine 9) antibody is then incubated with cells overnight before being fixed with 3% paraformaldehyde, 0.25% glutaraldehyde, and 0.25% Triton-X100, washed, and blocked with 5% milk in tris-buffered saline with 0.1% Tween-20 (TBST). Following a secondary antibody addition and plate washing, DELFIA reagents are used to boost the signal. Using non-linear regression analysis and a sigmoidal dose-response (variable slope) equation, the europium counts are normalized to the protein concentration, and the IC50 value for each inhibitor is determined[1].

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Prostate Cancer Cell Proliferation Assay (SRB Method): PC-3 or LNCaP cells were seeded in 96-well plates at a density of 4×10³ cells/well and cultured overnight at 37°C with 5% CO2. AT7867 was added at concentrations ranging from 0.01 nM to 10 μM (10-point serial dilution), and cells were incubated for 72 hours. After incubation, cells were fixed with 10% trichloroacetic acid for 1 hour, stained with 0.4% sulforhodamine B (SRB) for 30 minutes, and washed with 1% acetic acid. Bound SRB was dissolved in 10 mM Tris base, and absorbance was measured at 510 nm. IC50 was defined as the concentration of AT7867 that inhibited proliferation by 50% relative to vehicle control [1]
- Ovarian Cancer Apoptosis Assay (Annexin V-FITC/PI Staining): SKOV3 cells were seeded in 6-well plates at 2×10⁵ cells/well and treated with AT7867 (0.1-5 μM) for 48 hours. Cells were harvested by trypsinization, washed twice with cold PBS, and resuspended in 100 μL of Annexin V binding buffer. Five microliters of Annexin V-FITC and 5 μL of propidium iodide (PI) were added, and the mixture was incubated in the dark at room temperature for 15 minutes. Apoptotic cells were analyzed via flow cytometry within 1 hour, with early apoptosis defined as Annexin V-positive/PI-negative and late apoptosis as Annexin V-positive/PI-positive [1]
- Western Blot Analysis (Cellular): Cells were treated with AT7867 (0.1-5 μM) for 24 hours, then lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was determined using a BCA assay kit. Equal amounts of protein (30 μg per lane) were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 1 hour at room temperature, then incubated overnight at 4°C with primary antibodies against phospho-Akt (Ser473, Thr308), total Akt, phospho-p70 S6K (Thr389), phospho-S6 (Ser235/236), cleaved caspase-3, or β-actin. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies for 1 hour, and protein bands were visualized using an ECL detection system. Band intensity was quantified via ImageJ software [1]

Mice: Male athymic BALB/c mice (nu/nu) are used. BALB/c mice receive a single dose of AT7867 at 5 mg/kg intravenously (i.v.) and 20 mg/kg orally (p.o.). At each of the following time points—0.083, 0.167, 0.33, 0.67, 1, 2, 4, 6, 16 and 24 hours after an intravenous dose—as well as at 0.25, 0.5, 1, 2, 4, 6 and 24 hours after an oral dose—plasma samples are taken from duplicate animals. Mice are bled via cardiac puncture, and after all blood samples are centrifuged to obtain plasma, the plasma is frozen at -20°C until analysis. All plasma samples are prepared for bioanalysis using acetonitrile-containing internal standard protein precipitation. A standard calibration line made with AT7867 is used to quantify sample extracts, and an inhibitor-specific liquid chromatography tandem mass spectrometry (LC-MS/MS) technique is used for this. The parameters of pharmacokinetics are established.

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Prostate Cancer Xenograft Model (PC-3): Female nude mice (6-8 weeks old, n=6 per group) were subcutaneously injected with 2×10⁶ PC-3 cells (suspended in 100 μL of PBS + 50% Matrigel) into the right hind flank. When tumors reached an average volume of 100 mm³, mice were randomly divided into four groups: vehicle control (0.5% carboxymethyl cellulose sodium + 0.1% Tween 80), AT7867 25 mg/kg, AT7867 50 mg/kg, and AT7867 25 mg/kg + docetaxel 10 mg/kg. AT7867 was suspended in the vehicle and administered orally once daily for 21 days; docetaxel was dissolved in normal saline and administered intraperitoneally once weekly for 3 weeks. Tumor volume was measured every 3 days (volume = length × width² / 2), and body weight was recorded weekly [1]
- Ovarian Cancer Xenograft Model (SKOV3): Female nude mice (6-8 weeks old, n=5 per group) were subcutaneously injected with 3×10⁶ SKOV3 cells (in 100 μL of PBS + 50% Matrigel) into the left flank. When tumors reached ~120 mm³, mice were assigned to two groups: vehicle control (0.5% carboxymethyl cellulose sodium + 0.1% Tween 80) and AT7867 50 mg/kg. AT7867 was suspended in the vehicle and administered orally once daily for 14 days. At the end of the experiment, mice were euthanized, tumors were excised and weighed, and tumor lysates were prepared for Western blot analysis [1]

In male Sprague-Dawley rats, AT7867 was administered via two routes: intravenous (iv) 5 mg/kg and oral (po) 20 mg/kg. After intravenous administration, the plasma concentration-time curve conformed to a two-compartment model, with a terminal half-life (t1/2β) of 4.8 h, a steady-state volume of distribution (Vdss) of 2.6 L/kg, and a total clearance (CL) of 0.5 L/h/kg. After oral administration, the peak plasma concentration (Cmax) was 2.1 μg/mL, the time to peak concentration (Tmax) was 1.8 h, and the oral bioavailability (F) was 36% [1]. In vitro plasma protein binding studies using equilibrium dialysis showed that AT7867 had a high affinity for plasma proteins: 94% in human plasma, 92% in rat plasma, and 90% in canine plasma. In all tested species, the free fraction was < 10% [1] - In vitro metabolic studies using human liver microsomes showed that AT7867 was metabolized in a NADPH-dependent manner into three minor metabolites (M1-M3), of which CYP3A4 contributed approximately 65% of the total metabolism (confirmed by specific CYP3A4 inhibitors) [1]

In a 28-day repeated-dose toxicity study, male and female Sprague-Dawley rats were orally administered AT7867 at doses of 10 mg/kg, 25 mg/kg, and 50 mg/kg, respectively, once daily. In the 50 mg/kg dose group, both male and female rats experienced a 10% decrease in body weight, a 1.6-fold increase in serum ALT (alanine aminotransferase) levels compared to the control group, and histopathological examination revealed mild vacuolation of hepatocytes. No significant toxicity was observed at doses of 10 mg/kg or 25 mg/kg (no weight loss, no abnormal liver enzymes) [1]
- In the SKOV3 ovarian cancer xenograft model, administration of AT7867 at a dose of 50 mg/kg (oral, for 14 days) did not cause significant changes in weight or significant pathological abnormalities in major organs (liver, kidney, heart, lungs) [1]
- In vitro cytotoxicity assays in normal human peripheral blood mononuclear cells (PBMCs) showed that AT7867 had a CC50 of 14 μM and a therapeutic index (TI = CC50/IC50) of 50 (IC50 = 0.28 μM compared to PC-3 cells) [1]

[1]. AT7867 is a potent and oral inhibitor of AKT and p70 S6 kinase that induces pharmacodynamic changes and inhibits human tumor xenograft growth. Mol Cancer Ther, 2010, 9(5), 1100-1110.

AT7867 belongs to the piperidine class of compounds and has two aryl substituents (4-chlorophenyl and 4-(pyrazol-4-yl)phenyl) at the 4 position. It is an EC 2.7.11.1 (nonspecific serine/threonine protein kinase) inhibitor and an antitumor drug. It belongs to the pyrazole, piperidine and monochlorobenzene classes of compounds.

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AT7867 is a potent oral dual inhibitor of Akt and p70 S6K for the treatment of solid tumors (e.g., prostate cancer, ovarian cancer, breast cancer) with dysregulated PI3K/mTOR signaling pathway[1].
- Preclinical studies have shown that AT7867 targets two key nodes (Akt and p70 S6K) in the PI3K/mTOR pathway, thereby avoiding the adaptive resistance common in single-target inhibitors (e.g., Akt-only inhibitors or p70 S6K-only inhibitors)[1].
- AT7867 has good oral bioavailability (36% in rats), and its plasma concentration remains higher than its in vitro IC50 value for more than 12 hours after oral administration, supporting preclinical once-daily dosing. Model [1] - In ovarian cancer models, AT7867 showed activity against both PTEN-deficient and PTEN wild-type subtypes, indicating its broad application prospects in the treatment of ovarian cancer [1]

C20H20N3CL

337.8459

337.134

C, 71.10; H, 5.97; Cl, 10.49; N, 12.44

857531-00-1

AT7867 dihydrochloride;1431697-86-7

11175137

White to off-white solid powder

1.2±0.1 g/cm3

530.2±50.0 °C at 760 mmHg

274.5±30.1 °C

0.0±1.4 mmHg at 25°C

1.616

4.41

2

2

3

24

394

0

ClC1C=CC(C2(CCNCC2)C2C=CC(C3=CNN=C3)=CC=2)=CC=1

LZMOSYUFVYJEPY-UHFFFAOYSA-N

InChI=1S/C20H20ClN3/c21-19-7-5-18(6-8-19)20(9-11-22-12-10-20)17-3-1-15(2-4-17)16-13-23-24-14-16/h1-8,13-14,22H,9-12H2,(H,23,24)

4-(4-(1H-pyrazol-4-yl)phenyl)-4-(4-chlorophenyl)piperidine

AT7867 dihydrochloride; AT7867 hydrochloride; AT7867 HCl; AT7867; AT 7867; AT-7867

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~68 mg/mL (201.3 mM)
Water: <1 mg/mL
Ethanol: 5 mg/mL (14.8 mM)

Solubility in Formulation 1: ≥ 1 mg/mL (2.96 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1 mg/mL (2.96 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1 mg/mL (2.96 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 15% Captisol, pH 9: 10mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)